PROJECT SUMMARY Cells rely on spatially and temporally precise expression of proteins to carry out key biological processes. Thus, decoding the regulatory genome is a crucial step towards developing accurate models of cellular behavior. While transcriptional networks have been widely studied, post-transcriptional regulatory programs remain largely uncharacterized. Recently, we discovered the double-stranded RNA-binding protein TARBP2 acts as a global regulator of RNA stability (Goodarzi et al. Nature, 2014). However, the underlying molecular mechanisms through which this non-canonical TARBP2 pathway regulates RNA decay were unknown. Based on our observation that TARBP2 binds extensively to the intronic regions of its target transcripts, we hypothesized and successfully demonstrated that this decay pathway is located in the nucleus. We also showed that TARBP2 interacts with different sets of proteins in the nucleus and the cytoplasm. Interestingly, among the nucleus-specific interactions were key components of the RNA methyltransferase complex and the nuclear surveillance machinery. Our preliminary findings strongly support a model in which TARBP2 binding results in the recruitment of the RNA methyltransferase complex and the subsequent methylation of TARBP2- bound introns. Methylated introns, which remain unspliced, are then targeted to the RNA exosome for degradation through an interaction between TARBP2 and the nucleoprotein TPR. To assess the veracity of this model, we will perform nuclear RNA sequencing to measure intron retention in the presence and absence of different components of this pathway. First, we will assess the role of TARBP2 binding in RNA methylation and its impact on splicing. Then, we will search for the components of the nuclear surveillance machinery that degrade transcripts with TARBP2-bound introns. We will also perform epistasis experiments to establish the pathway structure for this process. At every step, in addition to whole- transcriptomic measurements, we will use reporter constructs and CRISPR-mediated genome editing to test the requirement and sufficiency of TARBP2 binding, RNA methylation, and splicing in RNA decay. The successful completion of this study will result in the characterization of a novel regulatory pathway that uses targeted RNA methylation and orchestrated intron retention to modulate RNA abundance. The study proposed here builds on our years of multidisciplinary research on post-transcriptional regulation of gene expression (Goodarzi et al, Cell 2015, 2016; Goodarzi et al, Nature 2012, 2014). Our preliminary results, which strongly support our proposed model, provide a strong foundation for the interdisciplinary approach outlined in this proposal. Given our strong background in computational and experimental biology, and the expertise and support provided by our collaborators at UCSF, we are ideally situated to tackle this project.